The objectives of this project are to (l) identify and purify polypeptide factors from rat liver that inhibit proliferation of hepatocytes or hepatocyte cell lines, determining whether there is only one inhibitor or a family of cytostatic but non-cytotoxic hepatic proliferation inhibitors (HPI). The aim will be to obtain sufficient quantities of HPI(s) to allow detailed studies on the mechanism of action of these growth modulator(s). (2) Characterize the specificity of HPI(s) for inhibiting the proliferation of various cell types including normal hepatocytes from rats of different ages, preneoplastic and neoplastic hepatocytes, and cells from other tissues or species. (3) Determine whether HPI(s) are produced uniquely by hepatocytes or also by other normal, preneoplastic, or neoplastic cells. Results obtained so far include (1) development of methodology for (a) high resolution fractionation of tissue extracts by fast protein/peptide liquid chromatography; (b) rapid assay, for cells maintained in 96-well microtiter plates, of the incorporation of tritiated thymidine into DNA. Cells are pulsed with [3-H]-thymidine, released from the substratum by trypsinization, and collected with water washes onto glass fiber filters with a multi-channel cell harvester. Experiments with inhibitors of protein, DNA, and RNA synthesis indicate that the radioactivity retained on the filters selectively measures DNA synthesis. (c) Rapid fluorometric assay for DNA contained in the chromatin of cells attached to the wells of microtiter plates. In this assay, fluorescence resulting from interaction of chromatin DNA with Hoechst 33342 is measured automatically by a reflected light fluorometer. This fluorescence is directly proportional to cell or nucleus number and can be used for normalizing the incorporation of [3-H]-thymidine into DNA described in (b), above. (2) Demonstration that certain ethanol-precipitable fractions from adult rat livers cause a dose-dependent inhibition of DNA synthesis as assayed by the above methods.